Abstract
Prokaryotic DNA
gyrase is a type II topoisomerase that can introduce (-) supercoils to the DNA
substrates with the hydrolysis of ATP. Because DNA gyrase only exists in
bacterial cells and is an essential enzyme to bacteria, it is possible to
identify inhibitors targeting DNA gyrase without affecting host human enzymes.
Additionally, DNA gyrase can form covalent enzyme-DNA complex intermediates.
This property makes gyrase an excellent bactericidal target for developing
antibiotics. Fluoroquinolones are among the most successful
antibiotics targeted to DNA gyrase; however, since fluoroquinolones have been
explored extensively in terms of improving spectrum and potency, and overcoming
resistance, the limits of what these compounds can provide might have been reached.
Therefore, it is necessary to identify new types of compounds targeting
DNA gyrase. One challenge is to develop screening assays to identify inhibitors
in small molecule libraries that may target DNA gyrase. Additionally, there is
not cell-based method for identifying inhibitors targeting DNA gyrase. Since
compounds identified by the in vitro biochemical methods may not enter into
cells and function as antimicrobial agents, cell-based methods are preferable.FIU scientists
have developed materials and methods for
transcription regulation via divergently coupled promoters and their use in the
preparation of cells, polynucleotides, and assays for identifying gyrase
inhibitors. In this invention, a first promotor is linked to a first gene, and
a second promoter (linked to a second gene) is divergently coupled to the first
promoter. Transcription of the linked second gene under the control of the
second promoter is inhibited by negative supercoiling of the second promoter. A
compound is identified as a gyrase inhibitor based on differential expression
of genes under the control of divergently coupled promoters in the presence of
the compound. A compound that is not a gyrase inhibitor cannot inhibit the expression
of the first gene and cannot prevent negative supercoiling of the second
promoter. The materials include plasmids and E. coli strains to
identify inhibitors targeting bacterial DNA gyrase.
Benefit
Provides cell-based assays for identifying inhibitors targeting DNA gyrase in small molecule libraries
Market Application
Identification/screening of antibiotics for drug repurposing and/or development
Brochure
